ABSTRACT & SPECIFIC AIMS The complexity and heterogeneity of cancer makes it an extremely challenging disease to treat. Many traditional chemotherapeutics are highly toxic to both normal and disease cells. Ideal therapeutics would be able to recognize and distinguish cancer cells from normal cells, and respond with precise therapeutic action. In many ways, the ultimate smart therapeutics are living cells, which are highly complex sensor-actuator devices. Certain immune cells, for example, have the ability to detect specific signals and in response can precisely execute complex behaviors, such as targeted cell killing. The overall goal of our center is to use the tools of synthetic biology - the emerging science of building or reprogramming biological systems -- to engineer cells or cell-like devices that can execute custom designed anti-cancer response programs. As a vision, one can imagine that an ideal anti-cancer device would have the following properties: the ability to detect tumor antigens and environmental signals with high discrimination, resistance to tumor inhibitory signals (ones that evade the immune system), the ability to chemotax specifically to the site of the tumor, and the ability to conditionally evoke spatially and temporally controlled responses that either deliver therapeutics, kill tumor cells or reshape and disrupt the tumor environment. Such specitic behaviors would yield a highly selective anti-tumor therapeutic response. Over the tirst five years of this center, we have been developing tools for custom rewiring cell signaling pathways and for assembling complex cell-like liposomes. We are now focusing on applying these tools and strategies towards engineering cell or cell-like devices that are precisely tuned to detect and destroy tumor cells and the tumor microenvironment. Our specitic aims are to: 1. Engineer immune cells (T-cells, NK cells) with significantly improved ability to selectively detect and kill tumor cells. Cytotoxic immune cells can be targeted towards cancer cells by engineering them to express artiticial chimeric antigen receptors (CARs). Current data from adoptive immunotherapy clinical trials, however, indicates that such engineered cells, while potent in their anti-cancer effects, can have serious off-target side effects, reacting with normal cells that express low levels of a tumor associated antigen. To solve this critical problem, we are working to engineer synthetic control signaling circuits in CAR-expressing cells, which could tune and dynamically modulate their response protile for optimized therapeutic function with minimal toxicity. 2. Engineer neutrophils as therapeutic or diagnostic search & delivery platform Neutrophils are extraordinarily effective at chemotaxis. Since delivery of therapeutic or imaging agents to specific sites in the body is a major challenge In the treatment of cancer, we are Interested harnessing neutrophils as a search and delivery platform. We are engineering neutrophils in which chemotaxis is now directed towards novel disease related signals. We are also engineering these cells to carry potentially therapeutic cargos. 3. Engineer anti-cancer immune cells that are resistant to tumor evasion and that disrupt the tumor microenvironment Tumors often produce a favorable microenvironment that includes production of signals that suppress cytotoxic immune cells. We will attempt to engineer anti-cancer immune cells that are resistant to these evasive effects and that disrupt the pro-tumor environment. 4. Produce smart vesicles that can execute a therapeutic oncolytic program We have recently invented the method of microtiuidic jetting - a unique method that permits the assembly of liposomes with complex molecular components. Thus this methods gives us the ability to construct cell-like structures. We are using this method to construct an oncolytic device, which will deliver a combination of a plasmid and regulatory protein that will only replicate and express a cytotoxic gene in tumor (p53 deticient) cells.